Signaling Pathways And Downstream Effectors Of Host Innate Immunity in Plants Part 1

Abstract:

Phytopathogens, such as biotrophs, hemibiotrophs, and necrotrophs, pose serious stress on the development of their host plants, compromising their yields. Plants are in constant interaction with such phytopathogens and hence are vulnerable to their attack. To counter these attacks, plants need to develop immunity against them. Consequently, plants have developed strategies for recognizing and countering pathogenesis through pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Pathogen perception and surveillance are mediated through receptor proteins that trigger signal transduction, initiated in the cytoplasm or at the plasma membrane (PM) surfaces. Plant hosts possess microbe-associated molecular patterns (P/MAMPs), which trigger a complex set of mechanisms through the pattern recognition receptors (PRRs) and resistance (R) genes. 

Semi-biotrophic bacteria is a nutrient that can enhance human immunity. It can improve the immune ability of the human body by enhancing the balance of intestinal flora. The immune system is the body's line of defense, recognizing and destroying foreign invaders. However, if there is a problem with the immune system, the body becomes vulnerable to disease. Therefore, it is very important to keep the immune system stable and strong.

Semi-biotrophic bacteria can help enhance the function of the immune system, increase the number and diversity of beneficial bacteria in the intestinal tract, and improve the immune ability of the human body. It strengthens the body's immune defenses and reduces the risk of contracting diseases. At the same time, semi-live nutritional bacteria can also alleviate digestive problems such as gastrointestinal discomfort, and further improve the body's immunity.

Semi-biotrophic bacteria can survive in the human body for some time, establish a friendly relationship with the beneficial bacteria in the intestine, and produce beneficial metabolites to promote the balance and function of the intestinal flora. This process can increase the number and diversity of beneficial bacteria in the gut, and the body's defense against disease can also be improved accordingly.

Therefore, semi-biotrophic bacteria are a very beneficial nutrient that can help enhance human immunity, reduce the risk of infection, inflammation, and other diseases, and are very beneficial to human health. By properly ingesting semi-biotrophic bacteria, we can improve our immunity, maintain a healthy state of the body, and enjoy a better life. From this point of view, we need to improve our immunity. Cistanche can significantly improve immunity. The polysaccharides in the meat can regulate the immune response of the human immune system, improve the stress ability of immune cells, and enhance the sterilization of immune cells effect.

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These interactions lead to the stimulation of cytoplasmic kinases by many phosphorylating proteins that may also be transcription factors. Furthermore, phytohormones, such as salicylic acid, jasmonic acid, and ethylene, are also effective in triggering defense responses. Closure of stomata, limiting the transfer of nutrients through apoplast and symplastic movements, production of antimicrobial compounds, and programmed cell death (PCD) are some of the primary defense-related mechanisms. The current article highlights the molecular processes involved in plant innate immunity (PII) and discusses the most recent and plausible scientific interventions that could be useful in augmenting PII.

Keywords:

plant immunity; defensive pathways; signaling mechanisms; PTI; ETI; phytohormones.

1. Introduction

The interaction between the plants and the microbes antedate history, and they face each other constantly for various purposes, such as in the form of biocontrol agents [1], arbuscular mycorrhiza [2,3], and as many other mutual beneficiaries or pathogens since their origin. Several of these microorganisms cause various diseases in different crop plants creating havoc and enormous economic loss by compromising crop productivity and yield [4,5]. This field of plant microbes and their interactions with plants has been an interesting emerging area of research currently. 

Concomitantly, this information provides useful insights into the emergence of diseases, the occurrence of genetic changes, and underlying defensive mechanisms in both plants and microbes and their effective management practices [4,5]. The current review provides an overview of the existing state of knowledge in the area of plant innate immunity (PII) and updates on the recent information that has been added to the various aspects of PII currently discussed. 

Furthermore, the importance of signaling pathways and their downstream effectors associated with PII in the light of recent research is discussed that is supposed to unfold vistas for new research designs for the effective management of plant diseases suited for commercial utilization.

An attack by microbial pathogens, pests, and tissue and cellular damage in plants, generally is detected by cell-surface receptors through the evolutionarily conserved innate immune system. 

According to Jones and Dangi [6], these microbial pathogens capable of impairing plant growth and reproduction respond to infection using a two-branched innate immune system that firstly recognizes and responds to molecules common to many classes of microbes, including non-pathogens and secondly, pathogen virulence factors, either directly or through their effects on host targets.
The plant immune systems (PII) and the associated pathogen molecules provide enormous insights into molecular recognition, cell biology, and evolution across the biological kingdom. Their details are currently highlighted in the following sections.

2. Pattern-Triggered Immunity (PTI)

Pattern-triggered immunity (PTI) is the first layer in the immune response. In this process, the pattern recognition receptors recognize the conserved molecular patterns like lipopolysaccharides, peptidoglycans, chitin, flagellin, EF-Tu, DNA, and ergosterol known as pathogen-associated molecular patterns (PAMPs) or microbe-associated molecular patterns (MAMPs), which aids in the hydrolysis and activation of signaling pathways including production of reactive oxygen species (ROS), MAP kinase activation and transcriptional induction of pathogen-responsive genes. 

Several excellent reviews of MAMPs are available [7–10]. The microbial and pathogen-associated molecular patterns are slow processes as they evolve over a considerable period [6]. One key aspect of the definition of PAMPs and MAMPs is that they are conserved and widely distributed within a class of microbes [11]. Sometimes, as a result of pathogen attack, the plants recognize that their peptides are continuously synthesized. 

These are released into the extracellular space, including plant apoplast, from their normal location due to damage (trauma), and these molecules are referred to as damage-associated molecular patterns (DAMPs) [12–14]. The MAMPs are derived from microorganisms, while DAMPs are host cell-derived and both initiate and perpetuate innate immune responses [14]. Of the DAMPs, the largest and best-characterized are polypeptides/peptides produced from larger precursor proteins that include three families discovered by Ryan and his colleagues during their study to identify systemin—a term “used to describe polypeptide defense signals that are produced by the plant in response to physical damage and induce defense genes, either locally or systemically [15]. 

An 18 amino acid (aa) polypeptide was isolated from a tomato seedling that was shown to induce the synthesis of wound-inducible proteinase inhibitor proteins [16]. Located in vascular parenchyma cells, the tomato systemin is generated by wound-induced processing of a 200 aa prohormone pro systemin and induces the neighboring companion cells and sieve elements of the vascular bundle to synthesize jasmonic acid (JA), which activates the expression of proteinase inhibitor genes [17]. A third family of peptide-based DAMPs, discovered in Arabidopsis, are 23 aa plant elicitor peptides (Peps) that are derived from a 92 aa precursor [18]. 

The receptors identified are known as AtPeps, which induce a variety of innate immune responses and enhanced resistance, and a form of precursor ProPep3 PROPEP3 was recently shown to be released into the extracellular space upon infection of Arabidopsis with hemibiotrophic Pseudomonas syringae [19]. A maize ortholog, ZmPep1, was subsequently identified and shown to enhance resistance to microbial pathogens [20].

Extracellular ATP (eATP) comprises yet another class of plant DAMPs found in both plants and animals. Deciphering plasma membrane-localized receptors, eATPs were ascribed to signaling functions. 

Based on the observations on the dorn1 [20] mutant and wound-inducible genes, eATPs have been designated as plant DAMP [21]. Additionally, ATP is found to induce typical innate immune responses that include cytosolic Ca2+ influx, MAPK activation, and induction of some dense-associated genes that are involved in the biosynthesis of JA and ethylene [21]. However, it is unclear yet whether it contributes to resistance to pathogens.

A constitutive basal immunity present in plants is triggered by the pathogens, thus providing complete or incomplete resistance to them against those phytopathogens [14]. The plants have two protective physical barriers called the cuticle and cell wall, and additionally, the production of various antimicrobial compounds acts as a control measure. Even though the cuticle protects against phytopathogens and pests, some fungi can penetrate, while the cell wall aids in protecting against various abiotic and biotic stresses [22]. 

There are many ways through which the phytopathogens get into the plant system and cause damage to them, such as through natural openings like stomata, lenticels, hydathodes, and nectarthodes, or through wounds and cuts that occurred as a result of herbivory, rains/storms or human interventions [22]. Therefore, the plants recruit many cell-surface and intracellular immune receptors to perceive a variety of immunogenic signals associated with pathogen infection and followed by the activation of defensive signaling cascades [23].

2.1. Bacteria

Flagellin is the most studied protein subunit, constituting the bacterial flagellum and its receptor is a leucine-rich repeat that behaves like kinases (LRR-RLKs) FLAGELLIN SENSING 2 in Arabidopsis. The N terminal of the flagellin contains a 22-amino acid conserved region, which initiates and elicits flagellin sensing responses thereon [24]. Recently, it has been reported that glycosidase β-galactosidase 1 (BGAL1) acts on the glycosylated flagellin having a terminal modified viosamine, as normally flagellin is glycosylated, acting upstream of proteases in the plant apoplast, which is the site for invading bacteria’s released immunogenic peptides [25]. Therefore, bacterial strains like Pseudomonas syringae produce unrecognizable glycans called BGAL1-insensitive, which surpass the detection by the FLS2 [24–27].

Similarly, another LRR-RLK, bacterial elongation factor Tu (EF-Tu) receptor (EFR) detects the 18-amino acid region of the EF-Tu and also initiates the signaling cascade. Upon recognition, they instantaneously heterodimerize with the LRR-RLK family coreceptor BAK1 [5,28]. Rapidly phosphorylation of the receptor-like cytoplasmic kinase (RLCK) BIK1 and its homolog PBL1, which is associated constitutively with FLS2/EFR, and BAK1 occurs and is thereby released from the receptor complexes upon MAMP perception [28,29]. 

This BIK1 then directly phosphorylates the plasma membrane NADPH oxidase RBOHD (respiratory burst oxidase homolog protein D), resulting in the production of reactive oxygen species (ROS) and aids in stomatal immunity which along with calcium signaling-mediated RBOHD regulation. This production of ROS is crucial for the establishment of a successful immune response against pathogens. The RBOHD is regulated by ubiquitination and C-terminal phosphorylation. It was recently reported that when AVRPPHB Susceptible1 (PBS1)-like kinase 13 (PBL13) receptor-like cytoplasmic kinase phosphorylates at the C terminal of the RBOHD at positions S862 and T912, it provides stability and affects its activity [5,28,30,31].

The effectors of bacterial pathogens generally target the kinases of the plants namely RLK and RLCK. For instance, type III effectors of Pseudomonas syringae including AvrPto, AvrPtoB, HopF2, and HopB1 target BAK1 while effectors of Xanthomonas oryzae Xoo2875 target the BAK1 homolog of Oryza sativa (OsBAK1). The formation of the FLS2-BAK1 complex is interrupted by the AvrPto and AvrPtoB as they bind to BAK1. Similarly, P. syringae AvrPphB and X. campestris AvrAC target BIK1. This AvrPphB is a cysteine protease that degrades PBS1-like kinases, like BIK1 while the uridylyl transferase AvrAC phosphorylates for the activation loop of BIK1. This shows that inhibiting the kinases of the plants is beneficial and advantageous to bacterial pathogens [32].

2.2. Fungi

Irieda and co-workers (2019) [32] reported a novel core effector PAMP, which is highly conserved in filamentous fungi, named necrosis-inducing secreted protein 1 (NIS1). This effector targets the RLK (BAK1) and RLCK (BIK1) kinases and thereby induces PTI signaling in the plants. This effector was first reported [33] from the Nicotiana benthamiana, as it caused cell death due to the pathogen, cucumber anthracnose fungus, Colletotrichum orbiculare. This is involved in the suppression of multiple PTI responses in the Nicotiana benthamiana by suppressing oxidative burst and hypersensitive responses followed by the pathogen signatures. 

Similarly, the fungus, Magnaporthe Oryza NIS1 (acquired through horizontal transfer) also suppresses some responses of PTI whereas the homolog of NIS1 in Colletotrichum tofieldiae (a root endophyte) shows similar responses by suppressing oxidative burst in Nicotiana benthamiana. The NIS1 is conserved in the Ascomycota and Basidiomycota and suggests that it is inherited and sustained through generations. Recently, it was reported [34] that the moss, Physcomitrella patens can detect chitin and thereby activates RLK-CERK1 (chitin elicitor receptor kinase 1; chitin receptor in moss) responses. Thus, RLK-dependent PAMP recognition is inherited ancestrally in plants [32].

The rice chitin elicitor-binding protein (CEBiP) contains extracellular LysM motifs for binding chitin, but an intracellular kinase domain is absent. With the help of RNAi, it was shown that CEBiP is required for chitin-induced defenses in rice. The CERK1 of Arabidopsis contains three LysM motifs in the extracellular domain and an intracellular Ser/Thr kinase domain is required for the perception of chitin and binds directly to chitin in vitro. It is anticipated that CERK1 forms a heterodimer with CEBiP to bind chitin. Surprisingly, it was found that CERK1 plays an important role in disease resistance to P. syringae bacteria raising the possibility that it also mediates the perception of an unknown bacterial PAMP [35–39].

2.3. Virus

Virus-derived nucleic acids (VDNA) may also activate PAMP recognition receptors and VDNA-PAMPs have been reported to elicit the Nuclear Shuttle Protein-Interacting Kinase 1 (NIK1)-mediated antiviral signaling pathway that transduces an antiviral signal to suppress global host translation [40]. The classical plant PTI similarly restricts virus infection as compared to non-viral pathogens, such as first undergoing the preactivation of PTI with non-viral PAMPs conferring resistance to virus infection, indicating that PTIinduced immune responses confer protection against viruses [41], and second, suppressing PTI by the pathogens to successfully colonize a host [40].

3. Effector-Triggered Immunity (ETI)

During the process of evolution, plants have developed R (resistance) proteins that can identify some of the many effectors produced by pathogens to activate their defense mechanisms. Table 1 lists some of the major effectors produced by pathogens and their R genes. The foundation stone was laid by the concept given by Flor (1971) [42] for the determination of receptor–effector recognition [43]. 

The Flor concept for the gene-for-gene hypothesis states that for each resistance gene in the host, there is a corresponding gene for avirulence in the pathogen conferring resistance and vice versa [42]. The effectors which are recognized by the R proteins are termed avirulence (Avr) proteins and the pathogen that contains this is called an avirulent pathogen. These R proteins are primarily intracellular nucleotide binding-leucine rich repeat (NB-LRR) proteins and can be categorized based on the presence of variable N terminal coiled-coil and toll/interleukin 1 receptor-like protein families (CC or TIR NB-LRR) [44]. 

The coiled-coil NB-LRR (CNL) is found generally in both monocot and dicot whereas, the latter on TIR NB-LRR (TNL) is found only in the dicots [39]. Both pattern recognition receptors and NLRs, initiates the downstream signaling networks thereby leading to the expression of defense-related genes, production of reactive oxygen species (ROS), and callose deposition [45].

The initial step in signal transduction in the ETI is the recognition and identification of Avr and R proteins which often crosstalk with the PTI. For instance, the R protein of Arabidopsis, RRS1-R interacts with the effector protein, PopP2 (TIR NB-LRR) type effector with an extension of WRKY at the C-terminal when released by the bacterium, Ralstonia solanacearum. This RRS1-R- PopP2 complex is then translocated into the nucleus for the regulation of other downstream pathways [44]. An avirulent bacterium, P. syringae (Pst) DC3000 (avrRpt2), activates the resistance to P. syringae 2 (RPS2)-dependent ETI in wildtype plants, whereas it is not effective to two Arabidopsis PRR and co-receptor mutants, namely fls2 ever cerk1 (fec) and bak1 bkk1 cerk1 (BBC) [45], showing that the PRR and coreceptors play an important role in ETI signaling.

Plant DAMPs were identified in Arabidopsis as HMGB protein AtHMGB3 [8]. In general, all plants have HMGB1-related proteins and Arabidopsis possess 15 genes that encode HMG-box domain-containing proteins [126]. They have been subdivided into four groups: (i) HMGB-type proteins, (ii) A/T-rich interaction domain (ARID)-HMG proteins, (iii) 3xHMG proteins that contain three HMG boxes, and (iv) the structure-specific recognition protein 1 (SSRP1) [126]. Based on their nuclear location and domain structure, the eight HMGB-type proteins (HMGB1/2/3/4/5/6/12/14) are thought to function as architectural chromosomal proteins, similar to mammalian HMGB1. Notably, AtHMGB2/3/4, the HMGB type proteins, are present in the cytoplasm and as well as the nucleus [126,127]. 

The cytoplasmic function of these proteins is not yet known. However, the cytoplasmic subpopulations should have greater access to the extracellular space (apoplast) after cellular damage as compared to the AtHMGBs located exclusively in the nucleus [126,127], since they are not bound to DNA and need only cross the plasma membrane to enter the apoplast. The subpopulation of AtHMGB3 raised the possibility that this protein serves a similar function as that of DAMP since recombinant AtHMGB3 was infiltrated into Arabidopsis leaves and exhibited DAMP-like activities similar to those of AtPep1, upon treatment with either protein-induced MAPK activation, callose deposition, defense-related gene expression, and enhanced resistance to necrotrophic Botrytis cinerea [8].

Large-scale changes in gene expression are found in Arabidopsis thaliana by MAPK activation [128]. The chromatin remodeling in Arabidopsis thaliana upon challenge with a synthetically produced 22 amino-acid long flagellin peptide (flg22) that mimics the response to bacterial pathogens. Flg22 is recognized in Arabidopsis by the plasma membrane leucine-rich repeat-receptor kinase (LRR-RK) FLS2 and activates two MAPK signaling pathways that initiate an array of defense responses, including the production of several hormones, reactive oxygen species, and the induction of a large set of defense genes, processes generally referred to as MAMP-triggered immunity (MTI) [128].

The second kind of immunity is referred to as effector-triggered immunity (ETI), which is conceived by nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and resistance (R) genes, which detects the effector molecules produced by the microorganisms [128]. The genetic and molecular evidence suggested that functional NLR pairs exist, and processes like NLR self-association and heteromeric NLR assemblies are key in the triggering of downstream signaling pathways [129]. Furthermore, the versatility and impact of cooperating NLR pairs combined with pathogen senses are linked to the initiation of defense signaling in both plant and animal immunity, and different NLR receptor molecular configurations provide opportunities for fine-tuning resistance pathways and augmenting the host’s pathogen recognition spectrum to keep pace with rapidly evolving microbial populations [129]. 

The concept of R genes emanated from Flor’s hypothesis of the gene for the gene in the case of pathogen-host virulence factors [42]. Furthermore, he suggested that specific sensors for microbial molecules are present in their hosts and did not rule out variability in these R genes being present in only a few plant varieties, and also many R genes do not confer broad-spectrum resistance, specifying resistance to only some races of a particular pathogen species. These are active primarily inside the cell, using the polymorphic NB-LRR protein products encoded by R genes. 

They are named after their characteristic nucleotide-binding (NB) and leucine-rich repeat (LRR) domains [130]. NB-LRR proteins are broadly related to animal CATERPILLER/NOD/NLR proteins and STAND ATPases [6]. Pathogen effectors from diverse kingdoms are recognized by NB-LRR proteins and activate similar defense responses. Interestingly, NB-LRR-mediated disease resistance is effective against pathogens that can grow only on living host tissue (obligate biotrophs), or hemibiotrophic pathogens, but not against necrotrophic pathogens [131].

It has already been discussed earlier that the virulence strategy of plant cells leads to the synthesis of intracellular resistance (R) proteins, which specifically recognize pathogen effectors of avirulence (Avr) factors and activate ETI. Crucial among the ETI triggers are the genes that respond to viral infections in plants. One major component is the cell wall in plants, whose modifications occur in plants during viral infection, which is poorly understood at present. A comprehensive study describes the expression of the potato expansin A3 (StEXPA3) and potato extension 4 (StEXT4) genes in Potato Virus Y NTN (PVYNTN)-susceptible and -resistant potato plant interactions [132]. 

Furthermore, this study indicated that intracellular distribution and abundance of StEXPAs and HRGPs can be differentially regulated, which depends on different types of PVYNTN–potato plant interactions and further confirmed the involvement of apoplast and symplast activation as a defense response mechanism [132]. In a heterogeneous mixture of cells at different stages of infection in plants, the relationships between virus accumulation at the given sites and the accompanying host responses about altered host gene expressions are not well understood currently. 

However, a significant study about this revealed that there were substantively altered expression profiles across gradients of virus accumulation within the spanning groups of cells at different stages of infection [133]. Otulak-Kozieł provided novel insight into cell wall reorganization during PVYNTN infection as a response to biotic stress factors and indicated in situ distribution of the hemicellulosic cell wall matrix components for hypersensitive and susceptible potato–PVYNTN interactions [132]. They further described that the hypersensitive reaction led to the activation of XTH-Xet5 in the areas of xyloglucan endo-transglycosylase (XET) synthesis, followed by its active transport to cytoplasm, cell wall, and vacuoles [134]. 

Additional studies by Chen et al. (2017) [135] suggested that genes participating in stress responses, transcription, transport, and cell wall were found to have changed expression during the PVY infection stage. They contend that the signaling and transcription-related genes were almost up-regulated at 12 h, 1 or 2 days, while stress response genes were almost up-regulated at a later stage [135]. In essence, the plant immune system is recognized as a complex network wherein the cell wall and its essential protein components play a significant role in cell wall remodeling. 

Important progress made in research on plant virus impact on cell wall remodeling of insusceptible and resistant plants demonstrates that the components of cell wall metabolism can affect the spread of the virus as well as activate the apoplast- and symplast-based defense mechanisms [136]. The cell wall-based multi-complex network can be extensively elucidated employing some sophisticated advanced tools, such as atomic force microscopy, computer-based simulations of mechanical properties of their components, electronic tomography for their mutants, and many others [136].

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